The goal of Core A is to determine intracellular Ph (pHi) and study pHi regulation of all projects. The technique of whole spectrum fluorescence spectroscopy with dual wavelength indicator dyes is used to measure pH in vitro in biopsies, xenografts, and cultured cells (Wah1). This technique is unique, exceptionally accurate, and ideally suited to support the goals of this program. To identify the role of particular H+ exchangers in pHi regulation, specific inhibitors will be used in several melanoma cell lines. The major pH regulatory exchangers to be evaluated are (1) Na+/H+ transporter (NHE-1), (2) C1-HCO3-exchanger, 93) the H+-linked monocarboxylate transporter (MCT) and (4) Vacuolar (V-type) ATPase. Inhibitors of most specific H+ exchanger isoforms are already in hand. As additional corroboration of the validity of these studies, determination of pHi in large scale cultures will be performed by NMR spectroscopy (Glickson). In addition, Core A will quantify levels of the transporter proteins using flow cytometry in primary cultures of patient biopsies and in cultured cells grown at normal and low pH using isoform specific antibodies for further identification of the most promising transporters with the objective being to identify targets that are selective for melanoma cells at low pH 7.3. Various degrees of acidification during experiments with applied heat and/or pH regulation inhibitors are monitored in terms of effects on pHi to correlate with the work done in all projects. In support of Projects 1 & 2, xenograft microfragments will be studied ex vivo to measure pH, under various conditions. This will enable predictions to be made with respect to potential treatment effectiveness, window of opportunity for optimal combinations, and optimal timing with sequential approaches to therapy. These predictions can then be validated in melanoma xenografts in vivo. The contribution of pHi before, during, and after hyperthermia will be correlated with apoptosis and cell survival (Project 3) as a function of acute acidification and/or exposure to inhibitors of H+ exchange during hyperthermia. Support of Project 3 will also entail determination of whether regulation of apoptosis by heat shock proteins is influenced by Ph. Support of Project 4 will determine pH, in association with identification of signaling factors controlling DNA replication and induction of the S-phase checkpoint in the presence or absence of hyperthermia. Experiments devoted to measurement of pHi will be performed under conditions designed to match those in the projects. These measurements will be critical for interpretation of results from the projects and will assist in the development of therapeutic approaches that target tumor cells by taking advantage of the tumor microenvironment. This Core is fundamental to the entire Program Project.